Please use this identifier to cite or link to this item: https://hdl.handle.net/20.500.12202/3707
Title: Role of protein factors in initiation of translation in eukaryotic cells
Authors: Chaudhuri, Jayanta
Keywords: Molecular biology.
Cellular biology.
Issue Date: 1996
Publisher: ProQuest Dissertations & Theses
Citation: Source: Dissertation Abstracts International, Volume: 58-05, Section: B, page: 2266.;Advisors: Umadas Maitra.
Abstract: An obligatory step in initiation of translation in eukaryotic cells is the transfer of the initiator methionyl-tRNA (Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar}) as Met-tRNA{dollar}\sb{lcub}\rm f{rcub}\cdot{dollar}eIF2{dollar}\cdot{dollar}GTP ternary complex to the 40S ribosomal subunit to form a 40S preinitiation complex (40S{dollar}\cdot{dollar}Met-tRNA{dollar}\sb{lcub}\rm f{rcub}\cdot{dollar}eIF2{dollar}\cdot{dollar}GTP). The 40S preinitiation complex can then bind the 5{dollar}\sp\prime{dollar} end of mRNA to scan the message and locate the correct initiation AUG codon from which to begin polypeptide chain synthesis. The major focus of this thesis is to study the requirements for efficient in vitro formation of the 40S preinitiation complex and to identify and characterize the protein factors involved in the process. Towards that goal, we have developed an efficient in vitro translation initiation system that specifically measures the interaction of the initiator Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} with 40S ribosomal subunits prior to the binding of mRNA to form the 40S preinitiation complex. Using such a system, we observed that the binding of Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} to 40S ribosomal subunits required the participation of the 17 kDa translation initiation factor, eIF1A (formerly called eIF-4C). Furthermore, eIF1A alone mediated nearly quantitative transfer of Met-tRNA{dollar}\sb{lcub}\rm f{rcub}\cdot{dollar}eIF2{dollar}\cdot{dollar}GTP ternary complex to 40S ribosomal subunits. The 40S complex formed, however, was found to be free of eIF1A indicating that the role of eIF1A in the formation of the 40S initiation complex is not to stabilize binding of Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} to 40S ribosomes. In agreement with these results, we also observed that eIF1A was catalytically reutilized in the transfer of Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar} to 40S subunits to form the 40S preinitiation complex.;Further characterization of the eIF1A-mediated 40S preinitiation complex formation showed that addition of 60S ribosomal subunits to a preformed 40S preinitiation complex causes nearly complete disruption of the ribosomal preinitiation complex. If however, the 40S preinitiation complex was formed with 40S subunits that were preincubated with eIF3, the 40S preinitiation complex (40S{dollar}\cdot{dollar}eIF3{dollar}\cdot{dollar}Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar}eIF2{dollar}\cdot{dollar}GTP) formed was resistant to the disruptive effect of the 60S ribosomal subunits. These results suggest that while eIF1A alone is both necessary and sufficient to mediate the quantitative transfer of Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar}eIF2{dollar}\cdot{dollar}GTP ternary complex to islolated 40S subunits, in vivo when both 40S and 60S ribosomal subunits are present, eIF3, in addition to eIF1A, is required to form a stable 40S preinitiation complex.;To better understand the role of eIF3 in translation initiation, we have cloned and sequenced the cDNA encoding the 110 kDa subunit of eIF3. The amino acid sequence derived from the cDNA shows that the 110 kDa subunit is the mammalian homologue of yeast Prt1p, a protein that was previously shown to be a subunit of yeast eIF3 and is required for the transfer of Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar}eIF2{dollar}\cdot{dollar}GTP ternary complex to 40S subunits.;Finally, we show that initiation factors eIF2 and eIF5 specifically interact with each other to form an eIF2{dollar}\cdot{dollar}eIFS complex. The significance of eIF2{dollar}\cdot{dollar}eIFS complex formation in the overall mechanism of GTP hydrolysis in translation initiation is discussed.
URI: https://ezproxy.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9717861
https://hdl.handle.net/20.500.12202/3707
Appears in Collections:Albert Einstein College of Medicine: Doctoral Dissertations

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